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Oncolytic viruses are promising agents for cancer therapy because they selectively infect and kill tumor cells, and because they trigger immune responses that can boost anticancer immunity. Key to the latter process is the production of type I interferons (IFN-Is) that can turn noninflamed "cold" tumors into "hot" ones. Besides this desired anticancer effect, IFN-Is are antiviral and successful oncolytic virotherapy thus relies on tightly controlled IFN-I levels. This requires a profound understanding of when and how tumor cells induce IFN-I in response to specific viruses. In this study, we uncovered two key factors that augment IFN-I production in transformed human myeloid cells infected with a tumor-selective reovirus. Viral replication and IFN-alpha/beta receptor (IFNAR) signaling progressively reinforced the levels of IFN-I expressed by infected cells. Mechanistically, both augmented the activation of interferon regulatory factor 3, a key transcription factor for IFN beta expression. Our findings imply that reovirus-permissive tumor cells themselves are a major source of IFN-I expression. As tumors can perturb the IFNAR pathway for their own survival, reovirus-exposed IFNAR-unresponsive tumors may need additional therapeutic intervention to promote the secretion of sufficient IFN-I into the tumor microenvironment. Our increased understanding of the parameters that affect reovirus-induced IFN-I levels could aid in the design of tailored virus-based cancer therapies.

ABSTRACT New World alphaviruses of eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) are endemic in North and South America, and infect humans and equine through mosquitoes. In addition, these viruses are highly infectious when aerosolized, making them potential biowarfare and bioterrorism agents. Currently, no approved vaccines or drugs are available for prevention and treatment. Extensive studies have been carried out to understand molecular mechanisms of virulence among New World alphaviruses. This review will focus on virus-encoded, interferon antagonizing proteins which play major role in determination of virulence of New World alphaviruses. Understanding of molecular mechanism of these proteins will shed light on development of broad-acting antivirals against New World alphaviruses.

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Background: Down syndrome (DS; trisomy 21) is the most common chromosomal aberration, with an incidence of 1/700 live births, and presents with mental retardation, typical phenotype and a series of associated pathological conditions. Although overexpression of genes encoded on chromosome 21 was proposed to lead to the phenotype, this has not been fully addressed at the protein level. Aim: It was therefore the aim of our study to determine protein levels of several chromosome 21 gene products in fetal DS brain. We selected interferon (IFN) α/β receptor β chain (IFNAR2), glutamate receptor 5 (ionotropic kainate 1), h2 calponin, claudin-8 and oligodendrocyte transcription factor 1 for quantification in fetal brain of the early second trimester using immunoblotting. Results: We observed significant and about twofold overexpression of the 20-kD immunoreactive IFN receptor α/β band in the fetal cortex of DS brain (p = 0.0293), while the 70-kD immunoreactive band was overexpressed but did not reach statistical significance (p = 0.065). When data were normalized to the housekeeping protein β-actin, a significant increase in IFN receptor levels was observed. Levels of the aforementioned proteins were comparable between DS and controls. Conclusion: Overexpression of IFNAR2 in fetal DS brain may increase the responsiveness of DS cells, leading to altered neuronal excitability, neuronal growth and survival as well as cytoskeleton derangement.

The influence of interferon-beta (IFN-beta) dosing time on antiviral activity was investigated in ICR male mice under light-dark cycle conditions (lights on at 07:00, off at 19:00) with food and water available ad libitum. There was a significant dosing time-dependent change in 2',5'-oligoadenylate synthetase (2',5'-OAS) activities, as an index of antiviral activity, in liver at 12 h after IFN-beta (15 MIU/kg, i.v.) injection. IFN-beta-induced 2',5'-OAS activity was more potent after the drug injection during the late dark phase. The higher antiviral effect of IFN-beta was observed when the interferon-alpha/beta receptor (IFNAR) expression in the liver increased, and the lower effect was observed when its expression decreased. IFN-beta-induced fever was more serious after IFN-beta injection from the late dark phase to the early light phase. A significant dosing time-dependent change was demonstrated for plasma IFN-beta concentrations, which showed a higher level during the light phase and a lower level during the dark phase. The dosing time-dependent change of plasma IFN-beta concentrations was not associated with that of the antiviral effect or fever induced by IFN-beta. These results suggest that selecting the most suitable dosing time of IFN-beta, associated with the 24-h rhythm of IFNAR expression in the liver, may be important to increase effectively the antiviral activity of the drug in experimental and clinical situations.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.

Both exogenous interferon-γ (IFN-γ) and interferon-α/β (IFN-α/β) stimulate Clq mRNA synthesis in murine macrophages. Previous studies suggested that IFN-γ induced endogenous synthesis of IFN-α/β by...

Induction and modulation of type I interferon synthesis upon BTV infection Bluetongue virus (BTV) is an arthropod-borne virus belonging to the Reoviridae family. It causes an haemorrhagic disease in ruminants that induces important economic losses. BTV infection triggers type I interferon (IFN-α/β) synthesis in vivo and in vitro, which is crucial to mount an efficient cellular antiviral response. This event requires viral replication since a UV inactivate virus is unable to induce IFN-β synthesis. We also showed that RNA helicases RIG-I and MDA5 are both involved in IFN-α/β production upon BTV infection. As this response is deleterious for viral replication, most of viruses have developed strategies to circumvent IFN action. We found that the non structural NS3 protein of BTV is a potent IFN-α/β inhibitor., Le virus de la fièvre catarrhale ovine (FCO ; Bluetongue virus, BTV) est un arbovirus appartenant à la famille des Reoviridae. Il est responsable d’une maladie hémorragique chez les ruminants qui provoque d’importantes pertes économiques. L’infection par le BTV induit la production des interférons de type I (IFN-α/β) in vivo et in vitro, événement essentiel pour l’établissement d’une réponse cellulaire antivirale. Cette production requiert la réplication virale puisqu’un virus inactivé aux UV a perdu la capacité d’induire la synthèse d’IFN-β. Nous avons aussi pu démontrer que les ARN hélicases RIGI et MDA5 étaient impliquées dans la production d’IFN-α/β en réponse au BTV. Cette réponse étant délétère pour la multiplication virale, la plupart des virus ont développé des stratégies pour limiter l’action de l’interféron. Nous avons ainsi pu montrer que la protéine non structurale NS3 de BTV était un puissant antagoniste de la voie des IFN-α/β., Vitour Damien, Doceul Virginie, Chauveau Emilie, Lara Estelle, Zientara Stéphan. Induction et modulation de la synthèse d'interféron de type I par le BTV. In: Bulletin de l'Académie Vétérinaire de France tome 166 n°3, 2013. pp. 256-264.

Serum soluble interferon-alpha/beta receptor (sIFN-alpha/betaR) and high-sensitivity C-reactive protein (hs-CRP) levels were evaluated in the patients with gastrointestinal and hepatobiliary-pancreatic cancer. We compared the sensitivity and specificity of serum sIFN-alpha/betaR with that of serum hs-CRP and evaluated the two diagnostic parameters in combination. Serum sIFN-alpha/betaR levels were measured in 92 patients and 25 healthy individuals by enzyme-linked immunosorbent assay. The diagnoses were 37 cases of hepatocellular carcinoma, 17 cases of pancreatic cancer, 15 cases of colon cancer, 13 cases of biliary tract cancer, and 10 cases of gastric cancer. Serum levels of sIFN-alpha/betaR and hs-CRP were significantly higher in the patients than in healthy individuals (p0.05). The optimal cut-off values of sIFN-alpha/betaR and hs-CRP were 3600pg/ml and 0.5microg/ml, respectively. The sensitivity and specificity for these thresholds were 94.6% and 88.0%, whereas positive predictive and negative predictive values were 96.7% and 81.5%. These results suggest that a combination of serum sIFN-alpha/betaR and hs-CRP thresholds may be more reliable diagnostic parameter for gastrointestinal and hepatobiliary-pancreatic cancer.

Rickettsia prowazekii Madrid E established persistent infections in cultures of growing L-929 cells. Although some L-929 cells died, the cultures survived, remained infected with rickettsiae, and continued to grow. R. prowazekii Madrid E also induced interferon in L-929 cell cultures, and this interferon modulated rickettsial growth. Production of interferon (anti-viral activity) by cultures of R. prowazekii-infected L-929 cells was directly related to the initial rickettsial infection and was blocked by erythromycin. The media collected from R. prowazekii-infected L-929 cells suppressed not only the replication of vesicular stomatitis virus but also the growth of R. prowazekii in fresh L-929 cells. Both anti-viral and anti-rickettsial activities in the media were neutralized by antibodies against murine interferons-alpha and -beta, but not by antibodies against murine interferon-gamma. In addition, a commercial preparation of virus-induced interferons-alpha and -beta also suppressed rickettsial growth in L-929 cells. The combination of treating L-929 cells with this virus-induced interferon and infecting them with R. prowazekii killed some of the L-929 cells.

Nature 517, 89–93 (2015); doi:10.1038/nature13801In this Letter, author J.-L.C. was inadvertently missing one of their affiliations; they should also have been associated with affiliation number 2 (St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York 10065, USA). This has been corrected in the online versions of the paper.

In the recent paper by Takaoka et al. (2003), the authors demonstrate that interferons-alpha and -beta stimulate p53 expression but not p53 activation. The increase in p53 expression translates into significant enhancement of apoptosis and reduction of chemotherapeutic dosages in vitro to destroy tumor cells. Furthermore, viral infections are also modulated by p53 in collaboration with interferons-alpha and -beta. These observations are significant and may lead to new paradigms for therapy if the high doses of interferon necessary to obtain the effects in vitro can be combined with more active interferons, interferons with minimal side effects, and/or novel delivery systems to target interferons directly to tumors.

8 pages, 5 figures, 3 tables., Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-alpha/beta) down-regulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-gamma. Moreover, IFN-alpha/beta also decreased cell death in LPS/IFN-gamma-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-alpha/beta) against the apoptosis induced by treatment with LPS and IFN-gamma showed that type I IFNs were very effective when added up to 1 h after IFN-gamma/LPS stimulation. Addition of IFN-alpha/beta 4 h after stimulation with IFN-gamma/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-alpha/beta suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections., This work was supported by grant PM95-007 from CICYT, Spain.

e12503 Background: O6-methylguanine DNA methyltranferase (MGMT) is one of the main mechanisms of chemoresistance for alkylating agents in malignant gliomas. Recent studies have showed that glioma stem-like cells (GSCs) could be the main reason for tumor recurrence and chemoresistance. In this study, we aimed to explore the effects of interferon-α/β against MGMT-positive glioma stem-like cells, and to investigate whether interferon-α/β can enhance the efficiency of temozolomide (TMZ) and the possible mechanism Methods: The growth inhibition effect of TMZ, with interferon-α or interferon-β, against the MGMT-positive GSCs from human glioma cell lines U251 and SKMG-4 (U251G and SKMG-4G) was evaluated by using Cell Counting Kit-8 (CCK-8) assay in vitro, and in xenograft models. MGMT and NF-κB expression in the tumor samples were determined by RT-PCR and Western blot analysis. Results: Our results revealed that the anti-tumor activity of TMZ was significantly enhanced by combined using interferon-α/β in vitro. In xenograft models, the tumor growth inhibit rate (IR) of TMZ to SKMG-4G and U251G was 35.2%±2.28% and 16.7%±1.96%, respectively. When TMZ combined with interferon-α or interferon-β, the IR to SKMG-4G was 58.4%±4.34% and 63.4%±1.08%; and to U251G was 41.1%±8.66% and 44.5%±1.90%, respectively(P

We have examined the antiproliferative effect of murine interferon (MuIFN) alpha/beta on a primitive class of erythroid precursors, the burst-forming units (BFUe) which give rise to macroscopic colonies of erythroblasts in plasma clots. Cultures were examined respectively on day 9 and on day 15; in both instances there was a significant reduction in the number of erythropoietic bursts in cultures exposed to IFN. The effect of IFN was more pronounced when the cells were cultured in the presence of suboptimal concentrations of erythropoietin and it could to a significant extent be reversed by ouabain at 10(-15) M. The latter finding is in line with observations made by others that growth factors can act as IFN antagonists. A comparison of BFUe derived from mice of two different genotypes, BALB/c and C57BL/6, revealed no difference in relative sensitivity to the antiproliferative effect of IFN. This is in contrast with the previously described fivefold greater sensitivity of the BALB/c genotype when a more differentiated class of erythroid precursors, the colony-forming units or CFUe, are exposed to IFN. Since the BFUe, studied in the present work, are a more primitive class of erythroid precursors than the CFUe, it can be concluded that the genes modulating the difference in sensitivity to IFN action on erythroid precursors are only expressed after a certain stage of differentiation has been reached.

Interferon-α/β (IFN-α/β)-sensitive (FLC 745) and -resistant (FLC 3C18) Friend leukemia cells (FLC) were fairly refractory to the cytotoxicity of murine tumor necrosis factor (MuTNF) in vitro. Both lines became highly sensitive to mTNF when treated in combination with murine IFN-γ; when treated with IFN-α/β, only the FLC 745 became sensitive to MuTNF. Romeo et al.(9) have reported previously that an antiviral state was induced in both FLC lines by IFN-γ, but only in FLC 745 by IFN-α/β. The present results suggest that the sensitivity to the synergistic cytotoxic effect of IFN and TNF in these cells is correlated with the ability to be induced into an antiviral state.

Nonsensitized murine lymphocytes produced mouse interferon when cocultured with foreign and transformed cells. Neutralization tests revealed that this interferon was of the α/β type. The B lymphocyte appeared responsible for the bulk of the interferon production as determined by cell “panning,” complement-mediated depletion, and immunofluorescence experiments.

Normal human adult lymphocytes were cultured with IFNs (α, β, and γ) in different concentrations and at culture times. The cells', supernatants' beta 2-m were measured by enzyme immunoassay. It was found the both beta 2-m were increased dependent upon in the culture IFN doses. The increasing percentages of beta 2-m was different for each IFN. The maximal expression of the cells' beta 2-m was observed for 24-48 hours after IFN treatment, in the IFN, maximal expression of cells' beta 2-m was for 24 hours. In the case of IFN-γ-cells' beta 2-m, they increased higher in the period of 96-hour culture. The increasing rate of beta 2-m with IFN-γ was higher than the IFN-α, β. It is supposed that beta 2-m is a useful new marker against cells' active condition.

Summary Swiss mice immunized with formalin-inactivated encephalomyocarditis virus (EMCV) produced antibodies to EMCV and were protected when infected intraperitoneally with EMCV. Administration of a potent sheep anti-mouse interferon-α/β (IFN-α/β) globulin before reinfection did not alter the vaccine-induced protection. Reinfection was associated with a rapid disappearance of the virus at the site of injection and viraemia was not demonstrable. IFN was not detected in the peritoneum or serum. Furthermore, injection of anti-IFN globulin before, during or after vaccination did not modify the humoral response to vaccination or to reinfection. These results indicate that IFN-α/β does not play a significant role in the resistance of mice to reinfection with EMCV.

Two human cell lines (HEC-1, IFr) which are resistant to the anticellular and/or antiviral action of HuIFN-α or -β have been tested with respect to the anticellular and antiviral activities of HuIFN-γ. HEC-1 cells were totally resistant to the antiviral activity of the IFN-γ, with either EMC or VSV as challenge virus. Per antiviral unit HuIFN-γ exerted a much stronger inhibitory effect on the growth of the interferon-sensitive cell line RSa than did IFN-α. IFr cells were more resistant to the anticellular effect of IFN-γ than the wild-type cells (RSa), and HEC-1 cells were totally resistant to the anticellular effect of HuIFN-γ. 2-5A synthetase was present at a high basic level in untreated HEC-1 cells, as previously reported. This activity was not influenced by either HuIFN-β or -γ. Similar levels of 2-5A synthetase were induced by HuIFN-γ in IFr and RSa cells; however, this induction was 10 times lower than that obtained for HuIFN-β. A 2′-phosphodiesterase activity was detected in control extracts from RSa and was not induced by either HuIFN-β or -γ. There was no correlation between the amplitude of 2-5A synthetase induction and extent of anticellular effect of either HuIFN-γ in different cell lines or of different interferons in a given cell line. Finally, HEC-1 represents the first example of a human cell line which is resistant to all three types of interferon (HuIFN-α, -β, and -γ).